title: Loss of BRCA1 leads to an increase in epidermal growth factor receptor expression in mammary epithelial cells, and epidermal growth factor receptor inhibition prevents estrogen receptor-negative cancers in BRCA1-mutant mice.
abstract: BACKGROUND
  Women who carry a BRCA1 mutation typically develop "triple-negative" breast cancers (TNBC), defined by the absence of estrogen receptor (ER), progesterone receptor and Her2/neu. In contrast to ER-positive tumors, TNBCs frequently express high levels of epidermal growth factor receptor (EGFR). Previously, we found a disproportionate fraction of progenitor cells in BRCA1 mutation carriers with EGFR overexpression. Here we examine the role of EGFR in mammary epithelial cells (MECs) in the emergence of BRCA1-related tumors and as a potential target for the prevention of TNBC.
  
  
  METHODS
  Cultures of MECs were used to examine EGFR protein levels and promoter activity in response to BRCA1 suppression with inhibitory RNA. EGFR was assessed by immunoblot and immunofluorescence analysis, real-time reverse transcriptase-polymerase chain reaction assay (RT-PCR) and flow cytometry. Binding of epidermal growth factor (EGF) to subpopulations of MECs was examined by Scatchard analysis. The responsiveness of MECs to the EGFR inhibitor erlotinib was assessed in vitro in three-dimensional cultures and in vivo. Mouse mammary tumor virus-Cre recombinase (MMTV-Cre) BRCA1flox/flox p53⁺/⁻ mice were treated daily with erlotinib or vehicle control, and breast cancer-free survival was analyzed using the Kaplan-Meier method.
  
  
  RESULTS
  Inhibition of BRCA1 in MECs led to upregulation of EGFR with an inverse correlation of BRCA1 with cellular EGFR protein levels (r² = 0.87) and to an increase in cell surface-expressed EGFR. EGFR upregulation in response to BRCA1 suppression was mediated by transcriptional and posttranslational mechanisms. Aldehyde dehydrogenase 1 (ALDH1)-positive MECs expressed higher levels of EGFR than ALDH1-negative MECs and were expanded two- to threefold in the BRCA1-inhibited MEC population. All MECs were exquisitely sensitive to EGFR inhibition with erlotinib in vitro. EGFR inhibition in MMTV-Cre BRCA1flox/flox p53⁺/⁻ female mice starting at age 3 months increased disease-free survival from 256 days in the controls to 365 days in the erlotinib-treated cohort.
  
  
  CONCLUSIONS
  We propose that even partial loss of BRCA1 leads to an overall increase in EGFR expression in MECs and to an expansion of the highly EGFR-expressing, ALDH1-positive fraction. Increased EGFR expression may confer a growth advantage to MECs with loss of BRCA1 at the earliest stages of transformation. Employing EGFR inhibition with erlotinib specifically at this premalignant stage was effective in decreasing the incidence of ER-negative breast tumors in this mouse model.
authors.affiliation: Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston MA 02215, USA.
authors.forename: Laura N
authors.initials: LN
authors.lastname: Burga
authors.forename: Hai
authors.initials: H
authors.lastname: Hu
authors.forename: Ashish
authors.initials: A
authors.lastname: Juvekar
authors.forename: Nadine M
authors.initials: NM
authors.lastname: Tung
authors.forename: Susan L
authors.initials: SL
authors.lastname: Troyan
authors.forename: Erin W
authors.initials: EW
authors.lastname: Hofstatter
authors.forename: Gerburg M
authors.initials: GM
authors.lastname: Wulf
mesh_terms: D000080924:Aldehyde Dehydrogenase 1 Family; D000818:Animals; D019313:BRCA1 Protein / Q000378:metabolism*; D001940:Breast / Q000166:cytology / Q000378:metabolism*; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000069347:Erlotinib Hydrochloride; D005260:Female; D019398:Genes, BRCA1; D016158:Genes, p53; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000378:metabolism; D008325:Mammary Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism / Q000517:prevention & control*; D008324:Mammary Tumor Virus, Mouse; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D034622:RNA Interference; D011960:Receptors, Estrogen / Q000378:metabolism; D050697:Retinal Dehydrogenase / Q000378:metabolism
chemical_list: D019313:BRCA1 Protein; D007527:Isoenzymes; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D011960:Receptors, Estrogen; D004815:Epidermal Growth Factor; D000069347:Erlotinib Hydrochloride; D000080924:Aldehyde Dehydrogenase 1 Family; C510223:ALDH1A1 protein, human; C515028:ALDH1A1 protein, mouse; D050697:Retinal Dehydrogenase; D066246:ErbB Receptors